1. Field of the Invention
The present invention relates to the detection, identification and monitoring of submicron size particles. More particularly, the invention pertains to apparatus and methods for the sampling, measuring, characterizing, automated detection, identification, and monitoring of submicron size particles. Preferably, the present invention provides for the sampling, detection and identification of submicron size particles having a size range of from about 5 to about 1000 nanometers. Such particles include viruses and virus-like agents (such as, for example, prions, viral subunits, viral cores of delipidated viruses, plant viruses, etc.) in bioaerosols and fluids. Further examples include standard particles used for calibrating equipment, coated particles, metallic-core shelled particles, polymers, fluorescent microspheres, powders, nanoclusters, particles produced as a result of manufacturing processes, and other chemical and biological materials such as segmented nanometer size portions of bacteria.
2. Fields of Use of the Invention
Detection and identification of viruses without limiting the detection and identification to a particular family, genus and species and searching for viruses pathogenic to humans in a single environment is difficult.
The difficulty of detecting and monitoring a wide range of viruses also varies by environment, but perhaps a most troublesome environment involves combat conditions, such as a potential biological warfare (BW) threat environment. Notwithstanding the variation in virulence from virus to virus, in general the ingestion of 104 virions constitutes a significant threat to a soldier who breathes on the order of 1,000 liters (1 m3) of air per hour. Instruments are needed with sensitivities which enable detection of remote releases of biological agents in a field environment thereby providing early warning capabilities, allowing calculations for troop movements and wind patterns.
Additionally, it has been difficult to maintain a broad-spectrum system for the detection of viruses which are free from false negatives because of natural or artificial mutations. Consideration should be given to the high mutation rates of known viruses, the emergence of new viruses, such as the Ebola virus, and the potential for deliberate artificial mutations of viruses. Furthermore, there are virus-like infectious agents, such as prions, which are suspected of causing scrapie, “mad-cow disease” and Creutzfeldt-Jakob disease. These prions possess no DNA or RNA, and can withstand 8 MRads of ionizing radiation before losing infectiousness. Other virus-like infectious agents, such as satellites, possess no proteins.
In the detection and monitoring of viruses recognition should be given to false positives associated with background materials. Background includes biological debris which obscures the detection of the viruses by registering as a virus when a sample is analyzed. Analysis of viruses requires a very high degree of purification of those viruses to overcome background loading in order to avoid false positives. For example, a BW virus may be buried within loadings of other microorganisms which form biological debris having loading on a magnitude of 1010 larger than the threshold loading for the targeted virus itself.
Although methods that culture viruses can often be used to increase the virus over background, culture methods may be too slow for effective viral BW detection; furthermore, some important viruses cannot be easily cultured.
As set forth in U.S. Pat. Nos. 6,051,189 and 6,485,686 and U.S. patent application Ser. No. 09/662,788 filed on Sep. 15, 2000, assigned to the U.S. Government and herein incorporated by reference, viruses may also be extracted from an environment and concentrated to an extent that permits detection and monitoring of viruses, without culturing procedures. Generally, in the detection of small amounts of viruses in environmental or biological liquids, it is necessary to both enrich the concentration of viruses many orders of magnitude (i.e., greatly reduce the volume of liquid solubilizing the viruses) and accomplish removal of non-viral impurities. In the presence of non-viral impurities, even the most sensitive detection methods generally require virus concentrations on the order of 10 femtomoles/microliter or more in the sampled liquid to reliably detect the viruses.
Sampling for airborne viruses is generally accomplished by collecting airborne particles in liquid, using a process such as air scrubbing, or eluting from filter paper collectors into a liquid medium. Collection and subsequent separation and detection methods are affected by the adsorption of viruses into solids in aerosols and liquids.
In contrast, when sampling liquids for viruses, in many cases no special equipment or processes may be necessary in order to collect a sample; for example, in sampling blood and other body fluids for viruses, only a standard clinical hypodermic needle may be needed. For sampling of bodies of water or other conveniently accessible liquids, sample collection may not be an issue at all, and in such cases the term “collector” is often applied to what is, in reality, a virus extraction step (such as collection on a filter).
Rapid detection translates into protection for soldiers, more reliable and simplified strategic planning, and validation of other BW countermeasures. Previously known detection methods using biochemical reagents may often be impractical in the field, even for trained virologists. Additionally, reagent-intensive approaches, such as multiplex PCR, low-strigency nucleic acid hybridization, and polyclonal antibodies, may increase the incidence of false positives several hundred-fold, whether under highly idealized laboratory conditions or in the field. Additionally, the hypervariability, or rapid mutation, of viruses and emergence of new, uncatalogued viruses may preclude methods based on biochemical assays, such as PCR, immunoassay, and the like, from achieving broad-spectrum detection of all viruses regardless of identity, known or unknown, sequenced or unsequenced.
With respect to nano-size particles having a size range of from about 5 to about 1000 nanometers, the ability to manufacture virus-size or nano-size particles has resulted in the commercialization of new processing technologies and potential applications of nanoparticles. Likewise, particles produced under controlled manufacturing conditions may contain nano-size contaminants. Generally, for example, nano or ultrafine powders made of a wide range of metallic, non-metallic, ceramic and semiconducter materials with particle sizes as small as 5 nm are examples of nano-size particles. Mechanochemical processing technologies may, for example, use a conventional milling process and induce specific solid-state reactions to uniquely form separated nano-particles. Nano-size particles may also have optical properties with a wide application in the creation of new transparent optical papers and film production. Uniform particle size distribution can result in uniform film quality and small particle sizes that can lead to enhanced resolution. Nano-size particles also have application as coatings and microchip manufacturing. Nanometer crystallites may consist of organically functionalized, catalytically active metals such as platinum, palladium and silver or non-active noble metals such as gold. In catalytic processes such particles have extremely high surface areas and size-dependent chemical behavior. In these advanced materials the electronic, thermodynamic and chemical properties frequently depend upon their size, shape and surface composition for functionality. It is a major challenge to control the particle size, morphology and surface composition. It is also a major challenge to properly characterize the particle size and morphology after the particles are manufactured. A near real time ability to measure and characterize these particles would be helpful.
Another example of nano-size manufacturing is the production of ultrafine metal particles by evaporation of a metal from a liquid pool, entraining the metal atoms in a hot inert gas carrier and then rapidly mixing in a cold inert gas to cause nucleation and growth. Nearly uniform sizes with controlled diameters in the nanometer range have been produced using this method. Such particles are collected by expanding the gas stream and impinging the particles onto a surface or by scrubbing the particles out of the gas stream with a liquid spray containing a surfactant and collect them as a stable colloid. This method has been used to take advantage of the unique electrical and optical properties of the nanoparticles, as well as, the using processes to deposit nanometer particles for making ultrasmooth surfaces and mirrors. Nanoparticles have also been made from sodium/halide flames. SEM (scanning electron micrography) images have shown hexagonal and cubic nanoscale (40–50 nm) particles of tungsten-titanium composites. This method prevents agglomeration by allowing nucleation and growth of the particles until they reach a desired size and then coat them with an appropriate material before they agglomerate. In this way encapsulated core nanometer particles can be produced. The coating material is removed by heating under a vacuum to produce a resulting powder. The benefit of encapsulation can be to narrow the size distribution of the core particles and thus improve the particle properties.
New methods for depositing nanometer-size thin coatings onto tiny particles are being considered for use in a wide range of applications that include the manufacturing of safe and more convenient medicines such as used in asthma therapy. Pulsed laser deposition techniques have been used to coat glucocorticoids, which are a component of asthma treatments, with thin layers of a biodegradable polymer. Such coatings are thought to improve the rate of drug release and improve overall blood concentration. A method to measure the increase in the diameter of the nanoparticles after coating would be helpful.
The uses and application of nanoparticles is rapidly expanding. Ceramic nanoparticles may provide better resistance to scratching and corrosion of paints and coatings. Improved manufacturing of nanoparticles could lead to improved catalysts thereby leading to new and better pharmaceuticals and materials. Batteries may generate more power as a result of the increased surface areas with metallic or iron-polymers.
To adequately measure and characterize nano-size particles, scanning-probe microscopes with supersharp tips, nanomanipulators, nanotubes, inorganic-organic hybrids and smaller electronics and other advances have produced requirements to adequately measure and characterize these particles.
For example, one means for counting, measuring and characterizing nanometer particles is with the use of nano-size polystyrene particles that are used as size-markers for measuring the dimensions of biological structures. These particles are available from companies such as Bangs Laboratories, Inc. for several standard sizes. The company generally sizes a particle three times and reports the average of the results. A frequently used method to measure nanometer size particles is light scattering technology, which yields a nominal mean diameter with a coefficient of variation.
Nano-size contaminants have been found in manufacturing processes. Nanometer particles have been discovered in amorphous films of silicon and hydrogen for the use in solar panels by use of a scanning tunneling microscope. These nanometer particles (3–5 nm) were thought to form in the vapor and bond with the film. They degrade the ability of the film to convert light into electrical energy. Measuring these nano particles and characterizing their distribution could help determine a way to keep the particles from forming or reaching the film surface and thus improve the films.
Bacteria are completely different types of microorganisms than the viruses. Viruses are a magnitude smaller in size than bacteria. Bacteria are classified in their own scheme. They have cell walls or are organized into cellular components and generally are considered to be among the self-sustaining organisims. Viruses require a living cell to invade in their life cycle. The technology and processes disclosed in U.S. Pat. Nos. 6,051,189 and 6,485,686 and the abovementioned co-pending application capitalize on the size and physical properties of the viruses to separate, count and characterize them. There is sufficient information from this characterization to identify them and perform investigative studies. Bacteria are generally 0.5–1 microns wide and 2–3 microns long, and generally outside the physical ability of the apparatus disclosed in referenced U.S. Patents. Bacteria have, however, interesting features that are in the proper size range for apparatus disclosed in referenced U.S. Patents to characterize. For example, gram-negative bacteria, named because of their inability to retain crystal violet-iodine complex stain, have rigid surface appendages called “pili.” These “hair-like” structures are around 7 nm in diameter and vary in length, up to 25 nm for the longer flagellae, which are other nanometer-sized structures that can be attached to the surface of bacteria. The Pili are composed of structural protein sub-units called “pilins.” Some structures have only one structural protein unit, other Pili are more complex and have several. These Pili consist of a precise helical arrangement of one or more types of protein and as indicated may have different lengths for different bacteria. Choudhury, et.al (1999): Science 7 Aug. 1999 285:1061 and David Eisenberg: How chaperones protect virgin proteins. (Science 13 Aug 99 285:1021), discuss crystal complexes associated with pilin subunits. Cell lysis breaks the cell into components. Lysis can be achieved by changes in pH, temperature, sonic treatment or by chemical means. The optimum means for releasing the pili proteins has not been well established, but their organization and structure indicates that controlled heating over a range of 63–70C will facilitate release. The pili can then been treated as any nanometer particle, separated and counted. Different pili proteins for different bacteria species can be expected. Evidence in this manner suggests that IVDS can indeed see these virus-sized bacteria components and in this manner detect bacteria. Pili are also found on gram positive rods and round or cocci bacteria.